Production of hyaluronidase from a strain of streptomyces

ABSTRACT

A NOVEL HYALURONIDASE IS PRODUCED BY CULTIVATING A NEW STRAIN OF STREPTOMYCES HYALUROLYTICUS.

United States Patent Oihce 3,728,223

Patented Apr. 17, 1973 hyaluronidase of the bovine testicle or bacteria has the 3,728 223 d f t th t 't P O UCTION OF HYAIZURONIDASE FROM A e cc a 1 becomes unstable during purification.

STRAIN OF STREPTOMYCES Yasuyuki Kaneko, Takaichi Ohya, and Geni'chi Amano,

It is noted that heretofore there has not been found any hyaluronidase contained in microorganisms other than Aichi Prefecture, and Hitoshi Oiwm Saitama Prefecture, bacteria. The present inventors have searched non-path- Japan assignors to Amano seiyaku Kabushiki Kaisha, ogenic microorganisms as a source of the hyaluronidase N Nagoya, Japan and have discovered that new species belonging to Strep- No Drawing. Continuation of abandoned application Ser. tomyces, which are isolated from soil, can accumulate a No. 784,460, Dec. 17, 1968. This application Oct. 8, large amount of the stable hyaluronidase in a culture 1971, 137,851 broth. The new species belonging to Streptomyces is Us Cl 195 62 CL C073 7/028 9 Claims recorded as the hyaluronidase-producing Streptomyces sp. No. 8l-10, and is named as Streptomyces hyalurolyticus nov. sp. by the inventors. ABSTRACT OF THE DISCLOSURE The mycological characteristics of the Streptomyces A novel hyaluronidase is produced by cultivating a new hy ticus (Streptomyces 81-10) are Strain of mm Ce h alum, rims. listed as follows and identified by the methods disclosed W Y s y y in Bergey's Manual of Determinative Bacteriology, 7th

edition (1957) and The Actinomycetes, vol. II (1961) This application is a continuation-in-part application of by S A, W k an Ser. No. 784,460, filed Dec. 17, 1968 now abandoned.

This invention relates to the use of a new Streptomyces (I) MORPHOLOGICAL CHARACTERISTICS Hyalurolyticzts in the production of a novel hyaluronidase, (1) Spore bearing hyphae: Simple, no verticillate, and to a method for the preparation of the hyaluronidase sporaphores straight or partial flexous, no spirals. by cultivating the strain of the new Streptomyces hya- (2) Spores: Elliptical, 1.0 x 0.7g, surface smooth, prolurolyticus. 2 5 duce chain and more than ten spores.

Hyaluronidase is an enzyme for depolymerizing the (3) (i) Globular sporangia: None. (ii) Flagellatcd hyaluronic acid to produce a low molecular weight sugar spore: None. (iii) Sclerotia: None. derivative. It is well known that the enzyme is contained (4) Aerial mycelium: Produce. in the testicle secretion of mammals, and is called the (5) Vegetative myceliurn: Thinner than aerial mycespreading factor. Also K. Meyer et al, have separated an lium, no segmentation, no septum.

enzyme from the crystalline lens of an eye ball and also have found that an autolyzated product produced by the (H) CULTURAL CHARACTERISTICS action of Pneumococcus decreases the viscosty of u o- Unless special mention is made, the cultural characterpolysaccharides and depolymerizes them to produce a reistics are observed after ten days cultivation at 30 C. ducing sugar. The enzyme is named by K. Meyer et al. as The citation of color in various cultures is based upon the hyaluronidase which corresponds to the spreading Standard of Color published by Japan Color Research factor. Institute.

Medium Growth Aerial mycelium Soluble pigment (1) Nutrient agar Moderate, ivory yellow, luster, surface yeast-like. None Yellowish brown. (2) Sucrose-nitrate agar Moderate, surface yeast-like wrinkled (under) Scanty, whlte Do. '(3) Synthetic agar.-.. Moderate, white- Risvgerse brown white pale orange, Pale yellowish brown,

a e. (4) Glucose-asparagin agar. Moderate, whit-e thin Scanty, reverse whiteto pale orange Do. (5) Ca-malate agar scanty, colorless Scanty, white None. ,(6) Glucose-peptone agar- Modqi'lfiilil hdplle tyellowish brown yeast-like alter scanty, grayish white Dark yellowish brown.

wn e un er (7) Egg Agar Moderlliitle,d reverse cream to brown, surface Moderate white Brownish black.

wnn e (8) Potato plug Abundant, yellowish brown, umbonate wrinkled Moderate, reverse to grayish white Dark yellowish brown. (9) Carrot plug None None None.

(l0) Oatmeal agar Abundant, white flat Abundant, reverse white to pale orange Oamell (yellowish brown) and to pinkish white. (11) Tyrosine agar scanty, colorless. None or scanty, white Dark brown. (12) Gelatin stab Colorless or cream, crateniorm or stratiform, None l. Dark reddish brown.

liquefaction strong. (13) Milk Abundant, white or cream, surface ring, coagulascanty, white Dark yellow. tiion, peptonization slowly, Iitomus no reduct on.

(14) Cellulose media None None None. (15) Peptone solution... Abundant, membranous, reverse whlte to gray- .do Deep brown.

ish white, no turb1dic. (1e) Nutrient broth Abundant, membramuadull white, produce None or scanty Light brown.

mycelial mat, no turbidlc.

Tihs hyaluronidase is found widely in the skin, spleen, testicle and semen of animals, the venom of bees and (III) PHYSIOLOGICAL PROPERTIES snakes, and the connective tissue of tadpoles. It is also found as the exoenzyme of pathtogenic bacteria in lnlCl'O- (1) Aer bic or anaerboic: aeroblc. organisms such as Clostridium welchii, Streptococcus (2) Th t t r range for growth and optimum hemolyticus, Staphylococcus auras, Pneumococcus and temperatum; g-40 C, 27 30 C the like. Such an enzyme acts to regulate the tissue perme- (3) The PH range for growth and Optimum PH: 541 ability and the fertilization of animals, and should be evaluated based on the permeation of toxin secreted by (4) Utilization of carbon Sources: arabinose xylose bacteria, and the nutrition of humans. Such an enzyme, which is physiologically and pathologically important, is used clinically. A commercially available hyaluronidase glucose mannose fructose lactose sucrose inositol rhamnose raffinose salicin p is produced from the bovine testicle and therefore the promanmtol glycenn Starch dexmn muhn duction of hyaluronidase is limited by the source. Also the galactose maltose sorbitol Liquefaction of gelatin (proteolytic action): positive (strong).

(6) Coagulation and petonization of milk (proteolytic action): positive (strong).

(7) Hydrolysis of starch (amylolytic action): positive (strong).

(8) Utilization of cellulose (cellulolytic action): negative.

(9) Nitrate reduction: positive.

(10) Tyrosiase (melanin formation): positive.

(11) Chromogenic action: positive.

(12) Hemolysis: negative.

(13) Hydrogen sulfide production: positive.

(14) Catalyase production: negative.

(15) Indol production: negative.

(16) Extinct temperature: 55 C. (10 mins.).

As is obvious from the fact that the Streptomyces sp. No. 81-10 promotes the growth of hyphae, produces the vegetative mycelium having no segment and does not form the sporangiurn, it evidently belongs to the family Streptomycetaceae. Also, the Streptomyces sp. No. 81-

10 cannot grow at a temperature of above 50 C., produces ten or more spores which are connected with each other into a chain-like state and has aerial mycelium, and therefore the microorganism falls within the genus Streptomyces. Further, it is considered that the Streptomyces sp. No. 81-10 belongs to the A-II group according to the classification by Waksman, as it is a microbe of the chromogenic type which does not form the verticillate. This A-II group contains 69 species, but they are different from the Streptomyces sp. No. 81-10 in growth and color and in that the spore bearing hyphae does not form spirals. Of the 69 species, it is considered that the 5 Streptomyces tanashiensis, Streptomyces griseochromogenes, Streptomyces garyphalus, Streptomyces bikiniensis, and Streptomyces mirabilis are similar to the Streptomyces sp. No. 81-10, but they have been tested and proved dilferent from the Streptomyces sp. No. 81-10, as shown in following Table l.

TABLE 1 [Comparison of Streptomyces sp. No. 81-10 and several similar strains] St. tanashiemis Nitrate reduction. Optium pH 5.8-6.5 7.0-8.0. Carbon utilization Sucrose, rafiinose, Opposite.

inulin-, sorbitol+. Sucrose nitrate agar- G: grayish-yellow White.

St. griseochromogems Starch agar Form closed spirals. No spirals. Tyrosine agar G: orange colored- White.

- SP: none Dark brown Milk coagulation. None ap d. Sucrose-nitrate aga G: orange-cinnamon...-. White. Carbon uti1izat1on.. Inositol+, inulin-, Opposite.

salicin.

St. garyphelus Nutrient agar G: colorless- Cream colored.

AM: grayish-white White. Potato. AM: grayish-black Do. M11 SP: dark purple to Dark yellowishbrownish-purple. brown. Gelatin SP: becomes greenish- Reddish-brown.

brown when shaken. Ca-malate agar AM: form None. Glucose-asparagine AP: none Faint yellowishagar. brown.

St. bikim'ensia Nutrient agar AM: moderate None. Milk coagulation- None Rapid. Glucose-asparagine SP: light amber Faint yellowishagar. brown. Sucrose-nitrate agar- Superficial droplets No droplet.

amber-colored. Gelatin Slight liquefaction Strong liquefaction. Starch agar Hydrolysis slight Strong hydrolysis.

G1 abunda t Moderate.

TABLE 1'Continued NOTE.AbbreViati0ns2 SP soluble pigment.

G=growth; AM=aerial mycelium;

Taking into consideration the above mentioned factors and the fact that the Streptomyces sp. No. 81-10 produces the new hyaluronidase, it can be recognized that the Streptomyces sp. No. 81-10 is a new species, which is named as Streptomyces hyalurolyticus nov. sp., based on its physiological properties.

The Streptomyces hyalurolyticus nov. sp. was deposited at Fermentation Research Institute, Agency of Industrial Science and Technology, No. 8-1 Inage, Higashi-S- chrome, Chiba-shi, Chiba-ken, Japan, under number FERM-P-427 (and also at Institute of Applied Microbiology, Tokyo University, Japan, under number I.A.M. 18012).

As is well known, the Streptomyces changes its own properties when it is grown on the various media, and therefore it will be understood that this invention includes the use of the new species and artificially modified strains for producing the hyaluronidase.

The Streptomyces hyalurolyticus nov. sp. PERM-P- 427 can be cultivated on a medium, which is used for the preparation of hyaluronidase, by using conventional culture methods such as an aerobic solid culture, a submerged culture and the like. A preferred culture method is a shaking culture or submerged culture.

The culture medium contains glycerin, glucose, soluble starch, sucrose or dextrin as a carbon source, casamino acid, peptone, meat extract, malt extract, corn-steepliquor, de-fatted soybean powder, yeast extract, urea or ammonium salts as nitrogen source, various kinds of phosphates, sodium chloride or MgSO -7H O as an inorganic salt, and optionally defoamers and other additives. It is preferable to use soluble starch or dextrin as the carbon source, and yeast extract, casamino acid or (NHQ SO as the nitrogen source.

When the Streptomyces hyalurolyticus nov. sp. FERM- P-427 is inoculated on such a culture medium and then cultivated at a temperature of 27 to 30 C. for 48 to 96 hours under aeration, the production of hyaluronidase reaches a maximum. The pH value of the culture broth may be lowered depending on the culture medium used. but the Stretptomyces hyalurolyticus nov. sp. PERM-P- 427 is not affected by the growth and production of the new hyaluronidase. If calcium carbonate is added to a culture medium, the production of hyaluronidase may occasionally be lowered.

For obtaining a crude hyaluronidase, at first the cells are removed from the culture broth by means of a suitable procedure and then the resultant culture filtrate, or concentrated culture filtrate which is produced by concentrating the culture filtrate under reduced pressure, is treated by a solvent precipitation procedure or a salting out procedure.

The organic solvents used in this invention include ethyl alcohol, methyl alcohol and acetone. The salts used for the salting out procedure include ammonium sulfate and magnesium sulfate.

The crude hyaluronidase contains a large amount of a melanin type brown pigment which is produced by the Streptomyces hyalurolyticus nov. sp. FERM-P-427 and other coloring matters which are derived from the culture medium, and such pigment and coloring matters cannot tivity are 5.0 and 65-70 C. respectively. The stable pH be completely removed by the conventional purifying range during 24 hr. at 37 C. is from 4 to 10. This enzyme procedures, and the purified hyaluronidase cannot be reis remarkably stable against heat and no appreciable decovered in high yield. crease of activity takes place by heat treatment up to 75- A method for removing the coloring matters, thus 80 C. for 30 min. A purified enzyme solution can be purifying the crude hyaluronidase and obtaining the 5 stored in the refrigerator for more than one month, withenzyme in high yield, is illustrated below. The anionout any accompanying decrease of activity. In contrast exchange resins and the adsorbent synthetic resins used to testicular or pneumococcal hyaluronidase, -Hg++ and can be re-used by washing with acids and alkalis. Such Mn++ ions inhibit the enzyme. A comparison of enzyme resins need no bufIerization procedure. properties of testicular, bacterial and Streptomyces At first, an anion-exchange resin is washed with a 2 hyaluronidases is shown in Table 2 Normal solution of hydrochloric acid and a 2 Normal TABLE 2 Animal hyaluronidase Bacterial hyaluronidase New hyaluronidase Source Bovine testicle Clostridiu'm welchii, Streptococcus Streptomyces hyalurolytz'cus nov. sp.

hcmalytims, Staphylococcus aureus. TERM-P427. Substrate specificity Hyaluronic acid, chondroitin, chon- Hyaluronic acid, chondroitin Hyaluronic acid.

droitin sulfuric acid. Heat stability Heat treatment (at 48 0., for 30 min- Streptococcus heat treatment (at 45 Heat treatment (at 70 0., 30 minutes):

utes): 50% residual activity. 0., minutes): inactive. 100% residual activity. Do Heat treatment (at 100 0., for 5 min- Staphylococcus heat treatment (at Heat treatment (at 0., 30 minutes): utes): 80% residual activity. 55 C., 5 minutes): 10% residual 95% residual activity. Very stable.

activity.

pH stability. Broad range 1 up pH 8.0 pH 4.0-11.0 broad range. Optimum pH" 5.5-6.2 5.5452 5.0. Inhibitor Fe++, Cu Zn". Mn++, Hg.

1 Unstable.

solution of sodium hydroxide and then the resin is washed 30 From the above results the hyaluronidase produced by with water until the eflluent water has a pH value of 5 to Streptomyces hyalurolyticus nov. sp. FERM-P-427 is 6. The washed resin is charged into a column and then proved to be clearly different from that of bacteria and a solution containing the crude hyaluronidase is passed animal. This is the first case microbial hyaluronidase has through the column. The pH value of the resultant effluent been found in sources other than pathogenic bacteria. is varied from 5 to 9 and therefore the pH value is An assay of the hyaluronidase of this invention Was peradjusted to about 5.0 with a 1 Normal solution of hydroformed by Tolksdorfs innovated method in which hyaluchloric acid to the effluent. ronic acid was prepared from umbilical cords by Dorf- The efiluent is then passed through a layer of an adsorbmans method. A solution containing approximately 1 ent sysnthetic resin, which is charged into a column and mg./ml. of hyaluronic acid was prepared by dissolving treated with acid and alkali in the same manner as menthe hyaluronic acid in a 0.02 M McIlvaine buffer (pH tioned above, and the pH value of the resultant effiuent 5.0) containing 0.2 M NaCl. To perform the assay, 0.5 is adjusted to about 5.0 by adding the 1 Normal solution ml. of enzyme solution was mixed with 0.5 ml. of hyaluof hydrochloric acid to the effluent. ronic acid solution (solutions were previously prepared Finally, the efiluent is passed through a layer of a at 60 C.) and incubated at 60 C. for 30 minutes. At the bufferized, weak cation-exchange resin having a pH value end of this operation 4 ml. of acid horse serum diluted by of 4 t0 5 to absorb the hyaluronidase on the cation 40 fold with the above buffer were added quickly, and

exchange resin. The adsorbed hyaluronidase can be reexactly 10 minutes later the optical density was determined moved from the cation-exchange resin by dissolving it with with a spectrophotometer at 660 mu. Controls were used a 0.5 mol acetate buffer solution. The product is the puriwith inactivated enzyme solution (heated at 100 C., 10

fied colorless hyaluronidase. minutes) and performed by the same method. Under The anion-exchange resins used in this invention these conditions, 1T.R.U. (turbidity reducing unit), de-

include Duolite A 2, A-4, A-6 and A-7, which fined as the enzyme concentration reducing the turbidity are manufactured by Diamond Alkali Company, Amby half, was observed.

berlite and XE225 which is manufactured by Rohm This invention is illustrated by the following examples.

and Haas. The adsorbent synthetic resins used in EXAMPLE 1 this invention include Duolite E833 and Duolite ES-30 which are manufactured by Diamond Alkali Com- 100 of a Culture medium (P Containing 25% pany. The cation-exchange resins used include Duolite glucose, CaSflmiIlO acid, 0.5% da -n 025% ES-8O and Duolite CS-10l which are manufactured by yeast extract and 0.1% K2N'PO4 were Poured into a 500 Diamond Alkali Company, and Amberlite IRC -SO and ml. shaking flask. After sterilizing for 15 minutes at 120 A b lit XE 64 hi h are uf t d b R h d C., the culture medium was inoculated with Streptomyces Haas. In addition to such resins, it is noted that diethylhyallu'olyfl'clls P- FERMP-4l27 which Was p aminoethyl cellulose (DEAE-cellulose) can be used for lured 0D the Same medium for 48 hours at and purifying the crude hyaluronidase. then a shaking culture was conducted for 80 hours at 30 This enzyme preparation has the activity to depolymer- 65 C. The cells were removed by filtration. The activity of ize hyaluronic acid to oligosaccharide, which is detected hyalur nidase Contained in the culture filtrate was 13 by reduction of aniline hydrogen phthalate, but it does not The D Value Of the l e filtrate was adform N-acetylglucosamine and glucuronic acid. Also, such justed to 5.0 and was fractionated between 40% and 60% activity is demonstrated by breaking down a protein- Saturation with ammonium sulfate. The precipitate was turbidity forming power of hyaluronic acid and also by collected and dissolved with a suitable amount of water. decreasing a high viscosity of the substrate solution by the The solution was salted out and concentrated and the conenzyme. Quite different from testicular hyaluronidase, this centrate was dried under freezing. About 1.1 grams of the enzyme cannot decompose chondroitin and chondroitin crude hyaluronidase were obtained from 1 liter of the sulphate. culture filtrate. The activity of the powdered hyaluronidase The optimum pH and temperature of the enzyme ac 7 Was 10 T-R-U./mg.

7 EXAMPLE 2 10 liters of a culture medium (pH 7.2) containing 3.0% soluble starch, 0.5% pentose, 0.5% meat extract, 0.8% (NH SO 0.15% yeast extract and 0.1% K HPO were poured into each of four -liter jar-fermenters. After sterilizing for 15 minutes at 120 C., the culture medium was inoculated with 200 ml. of Streptomgzces hyalurolytz'cws nov. sp. FER-M-P-427 which was precultured on the same medium, and a shaking culture was conducted for 40 hours at 30 C. under agitation (300 r.p.m.) and aeration (15 liter per minute). The resultant culture broth was filtered to remove the cells thus obtaining 32 liters of culture filtrate containing hyaluronidase having an activity of T.R.U./ml. The culture filtrate was concentrated to 8 liters containing hyaluronidase having an activity of 94 T.R.U./ml. (pH=5.7). To the concentrate was added 28.0 liters of cold ethyl alcohol for producing a precipitate. The precipitate was washed with cold ethyl alcohol and then dried under reduced pressure and about 230 grams of a greyish white, crude enzyme powder were obtained. This crude enzyme has a hyaluronidase activity of 2.9 T.R.U./ mg.

A 10% aqueous solution of this crude enzyme was passed through a layer of Duolite A-2 resin at the rate of 500 mL/hr. which had been previously washed with an acid and alkali, and then the resin was washed with water. Since the pH value of the resultant effluent was varied to about 9.0, the pH value was adjusted to 5.0 by adding a 1 Normal solution of hydrochloric acid. This operation removed about 90% or more of the coloring matter contained in the solution of crude enzyme.

Then, the purified solution was passed through a layer of Duolite ES-33 resin, which had been previously washed with an acid and alkali, and then the resin was washed with water in the same manner as mentioned above. The pH value of the resultant efiiuent was varied to about 8.5 and it was adjusted to 5.0 by using a 1 Normal solution of hydrochloric acid. This operation removed about 9.0% or more of the coloring matter contained in the original solution of the crude enzyme. The enzyme was recovered at a yield of about 73% and the specific activity of the enzyme was increased to about 4 times the original activity.

Finally, the etfiuent was dialyzed in a 0.01 mol buffer solution (pH='5.0) containing acetic acid at 5 C. for 24 hours and was then passed through a layer of Duolite ES- 80 resin, which had been previously bufi'erized with the same buffer solution, for adsorbing the enzyme on the resin. The adsorbed enzyme was dissolved out from the resin by passing a 0.2 to 0.5 mole buifer solution containing acetic acid through the layer of resin. By carrying out this operation, all the excess coloring matter in the original solution was removed and a clear efiluent was obtained. The clear efiluent was dialyzed at a low temperature, concentrated under reduced pressure and dried under freezing for obtaining about 5 grams of the purified enzyme having a specific activity of 2297 T.R.U./mg. The enzyme was recovered at a yield of about 64%.

EXAMPLE 3 250 liters of the same culture medium as in Example 2 were poured into a 500 liter fermenter and the culture medium was inoculated with Streptomyces hyalurolyticus nov. sp. FERM-P-427 at C. for hours under agitation (200 r.p.m.) and aeration (1:1). About 1.3 kg. of a grayish white, crude enzyme powder were obtained. The crude emzyme had a hyaluronidase activity of 2.5 T.R.U./mg. 160 grams of the crude enzyme were dissolved in water to produce a 10% solution. The solution was passed through a layer of Duolite A-7 resin, which had been previously washed with an acid and alkali, and then washed with water at the flow rate of 500 ml./hr. The pH value of the resultant efiiuent was adjusted to 5.0 by adding a 1 Normal solution of hydrochloric acid to the effluent. Then the effiuent was passed through a layer of Duolite ES-33 resin which had been previously washed with an acid and alkali, and then washed with water. The pH value of the resultant eflluent was adjusted to 4.1 and the efiluent was dialyzed in a 0.05 mol bufier solution (pl-1:41) containing acetic acid at 5 C. The dialyzed efiluent was passed through a layer of Duolite CS-101 resin, which is a weak acidic cation-exchange resin and butlerized with a 0.05 mol buffer solution (pH=4.1) containing acetic acid, for adsorbing the hyaluronidase on the resin. The hyaluronidase was dissolved out from the resin by treating it with a 0.5 mol buffer solution (pH=6.0) containing acetic acid and the hyaluronidase-containing solution was dialyzed for 24 hours in a 0.01 mol buffer solution (pH=4.1). The dialyzed solution was passed through a layer of diethylamino ethyl cellulose, which had been previously bufferized with a 0.01 mol buffer solution, for adsorbing impurities on the cellulose and obtaining an efiluent which contains the hyaluronidase. The efiluent was dialyzed in a 0.005 mol buffer solution (pH=8.0) containing phosphoric acid and then the dialyzed efiiuent was passed through a layer of diethylaminoethyl cellulose, which had been previously buiferized with a 0.005 mol buffer solution, for adsorbing the hyaluronidase on the cellulose. The adsorbed hyaluronidase was dissolved out from said diethylaminoethyl cellulose by treating it with a 0.05 mol buffer solution containing phosphoric acid. By carrying out this operation, all the coloring matter was removed and a clear solution was obtained. The specific activity of the clear solution was increased to about times the original activity. The enzyme was recovered at a yield of about 30%.

What is claimed is:

1. Hyaluronidase, characterized in that it can depolymerize hyaluronic acid only, is stable against heat and does not lose its activity at a temperature of 70 C. to 80 C., has an optimum pH of 5.0 and optimum temperature of 60 C. for growth, and is stable at pH values ranging from 4.0 to 11.0.

2. A method for preparing the hyaluronidase of claim 1, comprising cultivating (Streptomyces hyalurolytz'cus nov. sp. FERMP427 on a suitable culture medium containing a carbon. source, a nitrogen source and an inorganic salt to produce a culture broth containing the hyaluronidase and recovering the hyaluronidase from the culture broth.

3. The method according to claim 2, wherein the culture broth is filtered tocollect a culture filtrate containing the hyaluronidase and the filtrate is concentrated, purified and dried to produce a substantially pure hyaluronidase.

4. The method according to claim 3, wherein the hyaluronidase is collected from the concentrated culture filtrate by precipitation with an organic solvent or salting out.

5. The method according to claim 2, wherein the carbon source is soluble starch or dextrin and the nitrogen source is yeast extract, casamino acid or ammonium sulfate.

6. The method according to claim 2, wherein the cultivation is conducted at a temperature of 27 to 30 C. for 48 to 96 hours.

7. The method according to claim 2, wherein the culture broth is filtered to collect a culture filtrate containing the hyaluronidase and the hyaluronidase is collected from the culture filtrate by precipitation with an organic solvent or salting out.

8. The method according to claim 2, wherein the cul- References Cited ture broth is filtered to collect a culture filtrate contain- Kaneko at a1 New Hyalumnidase Produced by Strep ing the hyaluronidase and the hyaluronidase is collected tomyces, Agr, i l, h l, 31, No. 12, 1967 (pp. from the culture filtrate by concentration and precipita- 1515 1516) tion with an organic solvent.

9. The method according to claim 2, wherein the 5 A, LOUIS MQNAQELL, P i E i culture broth is filtered to collect a culture filtrate con- NAFF Assistant Examiner taming the hyaluronldase and the hyaluromdase 1s collected from the culture filtrate by concentration and USCLX'R salting out. 10 l95-66 R 

